Globins such as hemoglobin and myoglobin are heme-containing oxygen carriers. By reversibly binding to oxygen in the presence of high oxygen concentrations and releasing it in regions or at times of low concentrations, these proteins considerably enhance the oxygen uptake rate of multicellular organisms over that allowed by mere passive diffusion. In unicellular organisms it is generally believed that the oxygen uptake rate is principally limited by the rate of transfer of dissolved oxygen in the environment or growth medium to the exterior cell surface. However, closer examination of cell structure reveals several potential diffusional barriers between environmental oxygen and the cytochromes where the oxygen finally undergoes reaction. For example, in gram negative bacteria, where the cytochromes are attached to the inside of the plasma membrane, the diffusing oxygen needs to cross transport barriers such as the cell wall, the outer membrane, the periplasmic space and the inner membrane before accepting electrons from metabolic reactions. In unicellular eucaryotes, where oxidative phosphorylation takes place in the mitochondria, there are further diffusional resistances. Small neutral molecules like oxygen are assumed to passively diffuse across these barriers; however, these barriers make a non-trivial contribution to the overall resistance to mass transfer to the actual reaction site and thus could be of significance under oxygen-limited conditions.
Physiological effects on growth due to depletion in dissolved oxygen levels has been demonstrated in the case of several organisms, including Escherichia coli, Saccharomyces cerevisiae, Pseudomonas strains, and Alcaligenes eutrophus. In E. coli for example, which has a very high affinity cytochrome, changes in dissolved oxygen tension leads to differential regulation of terminal oxidases, resulting in a decrease in the number of protons expelled per NADH molecule oxidized during aerobic respiration and, consequently, a possible adverse change in the stoichiometry of ATP biosynthesis. (Kranz et al., Journal of Bacteriology 158:1191-1194, 1984; Ingraham et al., Growth of the bacterial cell, Sinauer Associates, Inc. 1983, p. 147, both specifically incorporated herein.)
In addition to the respiratory oxygen requirement of aerobic organisms, oxygen-binding proteins have other potential applications as well, including, for example, the enhancement of particular oxidative transformations such as steroid conversions, vinegar production, biological waste treatment or enzymatic degradations, and in some steps in brewing or making distilled and fermented foods and beverages.
The filamentous bacterium, Vitreoscilla, a member of the Beggiatoa family, is a strict aerobe that is found in oxygen-poor environments such as stagnant ponds and decaying vegetable matter. Growth of the bacterium under hypoxic conditions results in a several-fold induction of synthesis of a homodimeric soluble heme protein (subunit MW 15,775) (Boerman et al., Control of heme content in Vitreoscilla by oxygen, Journal of General Applied Microbiology 28:35-42, 1982) which has a remarkable spectral (Webster, et al., Reduced nicotinamide adenine dinucleotide cytochrome o reductase associated with cytochrome o purified from Vitreoscilla, Journal of Biological Chemistry 249:4257-4260, 1974), structural (Wakabayashi, et al., Primary sequences of a dimeric bacterial hemoglobin from Vitreoscilla, Nature 322:481-483, 1986), and kinetic (Orii, et al., Photodissociation of oxygenated cytochrome o(s) (Vitreoscilla) and kinetic studies of reassociation, Journal of Biological Chemistry 261:2978-2986, 1986) homology with eucaryotic hemoglobins, and which is probably a true bacterial hemoglobin.
This protein was previously thought to be a cytochrome o, and it has been suggested to function in oxygen storage. However, biochemical discrepancies (Webster, et al., Oxygenated cytochrome o, Journal of Biological Chemistry 252:1834-1836, 1977) as well as the subsequent discovery of the true membrane-bound cytochromes o and d (DeMaio, et al., Spectral evidence for the existence of a second cytochrome o in whole cells of Vitreoscilla, Journal of Biological Chemistry 258:13768-13771, 1983; Webster et al., Federation Proceeding 44:678, 1985) led to further investigations of its spectral properties (Choc et al., Oxygenated intermediate and carbonyl species of cytochrome o (Vitreoscilla), Journal of Biological Chemistry 257: 865-869, 1982; Orii et al.. supra.) and the eventual determination of its probable amino acid sequences and partial homology with known hemoglobin sequences.
Although these articles disclose the conservation of most features characteristic of eucaryotic hemoglobins, and discuss, to some extent, the role or potential role it probably plays in oxygen utilization, none of these researchers had previously been able to isolate a portable DNA sequence capable of directing intracellular production of this bacterial hemoglobin or to create a recombinant-DNA method for its production. Additionally, there has been no published proof of any oxygen transport or other kinetic function for this protein in Vitreoscilla, or any suggestion in the literature of any benefit from the introduction of a bacterial hemoglobin in heterologous organisms. Moreover, there has been no suggestion that such an oxygen-binding protein would have a far-reaching range of applications.
Surprisingly, the present inventors have discovered a portable DNA sequence capable of directing the recombinant-DNA synthesis of a bacterial hemoglobin. The hemoglobin of the present invention, prepared by the recombinant-DNA methods set forth herein, will enable increased research into the growth of organisms in oxygen-poor environments. In addition, the oxygen-binding proteins of the present invention are useful in enhancing oxygen supply to cells or in other oxygen-utilizing processes, and for binding and separating oxygen from other fluids or gases. Furthermore, the oxygen-binding proteins of this invention are capable of increasing production of cells, or of proteins or metabolites normally made by a cell, or of natural or unnatural metabolites and proteins expressed in a cell via genetic manipulation. The proteins of this invention are also useful as selective markers in recombinant-DNA work, and have applications as diverse as enhancing certain oxygen-requiring steps in fermentation, enzymatic degradation, toxic chemical waste treatment, brewing, and particular oxidative reactions and transformations.
This invention also relates to certain DNA sequences which usually precede a gene in a DNA polymer and which provide a site for initiation of the transcription of that gene into mRNA. These are referred to as "promoter" sequences. Other DNA or RNA sequences, also usually but not necessarily "upstream" of a structural gene, bind proteins that determine the frequency or rate of transcription and/or translation initiation. These other sequences, including attenuators, enhancers, operators and the like, are referred to as "regulator" sequences. Thus, sequences which operate to determine whether the transcription and eventual expression of a gene will take place are collectively referred to as "promoter/regulator" DNA sequences.
The promoter/regulator sequences of genes are susceptible to enormous structural and functional variation, and in general, serve to regulate gene transcription in response to chemical and, sometimes, physical environmental conditions in and around the cell. Several generalized models for the action of promoter/regulator operation in gene transcription have been proposed. One model utilizes a repressor gene and a regulator sequence or operator sequence near the promoter of another gene. According to this model, transcription of the repressor sequence results in expression of a repressor protein which selectively binds to the operator sequence to effectively preclude transcription of the selected gene. An environmental signal, such as the increased concentration of a chemical acted upon by the protein product of the gene in question, may operatively inactivate the repressor protein, blocking its ability to bind to the operator sequence in a way which would interrupt transcription of the gene. Increased concentrations of a substrate could be seen as operating to induce synthesis of the protein which catalyzes its breakdown.
Another generalized model of operation of promoter/regulator sequences in the regulation of gene transcription suggests formation of an initially inactive form of repressor protein by the repressor DNA sequence. Such inactive form could not bind to an operator DNA sequence and disrupt selected gene transcription until it is combined with some other substance present in the cell, such as a compound which is the product of a reaction catalyzed by the protein coded for by the selected gene. Increased concentrations of such a reaction product in the cell would thus operate to repress the potential overproduction of proteins responsible for the product's synthesis. In these examples, the regulator protein functions to inhibit transcription. Other regulatory proteins have been described which potentiate or activate transcription of specific DNA sequences. Thus, there can be both positive and negative control proteins and corresponding regulatory DNA sequences.
Regulation of gene expression can also occur at the level of translation. For example, a regulator molecule could bind to a particular site on the messenger RNA, thus inhibiting or blocking translation.
Much of the genetic engineering activity to date has been oriented toward stably incorporating foreign DNA into cells, to provide not only a source of multiple copies of selected genes, but the large scale transcription and expression of commercially significant gene products.
The lactose ("lac") promoter/operator systems have been commonly used, for they are very controllable through the mode of action of the operator. When the operator is repressed, the DNA dependent RNA polymerase is completely prevented from binding and initiating transcription, thus effectively blocking promoter operability. This system can be derepressed by induction following the addition of a known inducer, such as isopropyl-beta-D-thiogalactoside (IPTG). The inducer causes the repressor protein to fall away so the RNA polymerase can function.
Cells transformed with plasmids carrying the lac promoter/operator system can be permitted to grow up to maximal density while in the repressed state through the omission of an inducer, such as IPTG, from the media. When a high level of cell density is achieved, the system can be derepressed by addition of inducer. The promoter is then free to initiate transcription and thus obtain expression of the gene products at yields commensurate with the promoter strength. However, certain of these inducible promoter systems are relatively weak and commercial or research productions using such systems do not urge the cell to generate maximum output.
In response to the need for microbial expression vehicles capable of producing desired products in higher yield, the tryptophan ("trp") promoter/operator system has become widely used. This system is one of several known systems with at least three times the strength of the lac promoter. However, it has the disadvantage of less promote control. The trp promoter is not inducible in the way the lac promoter is, namely, the bound repressor is not removed by induction. Instead, the system operates on a sort of feedback loop as described above. A system was devised whereby the attenuator region of the trp promoter/operator system was removed, with the resultant transformed cells being grown in tryptophan-rich media. This provided sufficient tryptophan to essentially completely repress the operator so that cell growth could proceed uninhibited by premature expression of any desired foreign proteins. When the culture reached appropriate growth levels, no additional tryptophan was supplied, resulting in mild tryptophan limitation, and, accordingly, derepression of the promoter with resultant expression of the desired protein gene insert. In application, this system has several disadvantages. For example, it is necessary to maintain high levels of tryptophan in the growth media to completely repress the promoter, and to permit the medium to become completely exhausted of tryptophan following full growth of the culture.
A hybrid system has been developed from the tryptophan and lactose promoter, wherein both promoters can be repressed by the lac repressor and both can be derepressed with IPTG. See De Boer et al., The tac promoter: A functional hybrid derived from the trp and lac promoters, Proc. Natl. Acad. Sci. USA, 80: 21-25, 1983. This system shares a disadvantage with the two discussed above, namely the required introduction of additional agents to a normal growth medium.
Another regulator/promoter system commonly used for expression of cloned proteins in E. coli is based on the P.sub.L promoter system from phage lambda. See Bernard and Helsinki, Methods in Enzymology, 68:482-492, 1979; Use of Lambda Phage Promoter P.sub.L to Promote Gene Expression In Hybrid Plasmid Cloning Vehicles. Induction of this promoter requires increase of culture temperature from 30.degree. C. to 42.degree. C. This system has the disadvantages of suboptimal growth rates at 30.degree. C. prior to induction and upsetting of cell metabolism by the temperature shift. Temperature shift effects on metabolism are discussed, for example, by Neidhart, et al., The Genetics And Regulation Of Heat-Shock Proteins, Annual Reviews of Genetics, 18:295-329, 1984.
There has been a need in the art for an economical, simple, highly controllable and efficient promoter/regulator system for subjecting the transcription of DNA sequences to selective regulation by external control at constant temperature. The present inventors have discovered such an expression system, which can switch from low to very high expression activity upon reduction of dissolved oxygen concentration in the medium. This reduction in dissolved oxygen level is easily implemented at high cell densities without the need for addition of any chemical to the growth medium to induce gene expression.